This study is designed to elucidate the molecular regulatory mechanism of dental root development. Despite its important physiological and biological functions, dental root development and its regulation are not well understood. The root development involves continuous interactions between the dental epithelium (Hertwig's Epithelial Root Sheath, HERS) and the cranial neural crest derived mesenchyme, hence the root is an excellent model to investigate tissue-tissue interactions in regulating organogenesis. Recent studies show that Bmp/Tgf-, Shh, Fgf, Wnt, and Nfic may be involved in regulating root development. We have discovered that Smad4-mediated Bmp/Tgf- signaling plays a crucial role in regulating root development. However, it is unclear whether the inductive signal(s) of root formation resides in the dental epithelium or mesenchyme and how the Bmp/Tgf- signaling network controls root development. In this study, we will carry out experiments to test the hypotheses that Smad4 mediated Bmp/Tgf- signaling and their downstream target genes, such as Fgf10, Wnt, Nfic, and Bmps/Tgf-s, are crucial for mediating tissue-tissue interactions and controlling the fate of HERS and dental mesenchymal cells during root development. We have proposed three Specific Aims. In Specific Aim 1, we will test the hypothesis that epithelial Smad4 is required for HERS and dental mesenchymal cell fate determination during root development. We will investigate Smad4- mediated Fgf10 signaling and its functional significance in the induction of root development. In Specific Aim 2, we will investigate the functional significance of the Smad4-mediated Bmp/Tgf- and Wnt signaling cascades in regulating the fate of the dental mesenchyme and root development. We will elucidate the molecular mechanism(s) by which signals from the dental mesenchyme control root development. We will investigate mesenchymal Smad4/Msx2 interaction and its role in regulating root development. In Specific Aim 3, we will investigate the biological significance of Nfic-mediated Bmp and/or Tgf- signaling during root development. We will elucidate the regulatory mechanism(s) that control mesenchymal-epithelial interaction during root development. Ultimately, this study will provide a better understanding of the molecular regulatory mechanism of root development. The knowledge gained from this study will serve as the foundation for stem cell mediated tooth regeneration. PUBLIC HEALTH RELEVANCE: Dental roots are crucial for oral health because they provide anchorage for our dentition, transmit occlusal force to the jaw bone and send bio-feedback to our central nervous system. Despite recent advancements in research on the regulation of early tooth development, we still have limited knowledge of the regulatory mechanism of root development. Our research program is designed to further our understanding of how aberrant growth factor signaling networks may adversely affect cell fate determination, tissue-tissue interactions and cause root development defects.